Antisense oligonucleotides (aso) for efficient and precise rna editing with endogenous adenosine deaminase acting on rna (adar)

Abstract

The present invention relates to a chemically modified oligonucleotide for use in site-directed A-to-I editing of a target RNA inside a cell with endogenous ADAR, comprising a sequence with a length of 11 to 100 nucleotides capable of binding to a target sequence in the target RNA, with a Central Base Triplet of 3 nucleotides with the central nucleotide opposite to the target adenosine in the target RNA, which is to be edited to an inosine, whereby the core sequence has the following Formula I:wherein Nu stands for a nucleotide having a sugar moiety which may be modified, the numbers below the nucleotide sequence designate the position of the nucleotides adjacent to the central nucleotide of the Central Base Triplet having the number 0 whereby the negative numbers designate the 5′ end and the positive number designate the 3′ end of the oligonucleotide and wherein a-j designate the nature of the linkage between the single nucleotides whereby at least linkages a, d, and e are phosphorothioate linkages and whereby at least 2 linkages are a phosphate linkage(s).

1 . A chemically modified oligonucleotide for use in site-directed A-to-I editing of a target RNA inside a cell with endogenous ADAR, comprising a sequence with a length of 11 to 100 nucleotides capable of binding to a target sequence in the target RNA, with a Central Base Triplet of 3 nucleotides with the central nucleotide opposite to the target adenosine in the target RNA, which is to be edited to an inosine, whereby the core sequence has the following Formula 
       
         
           
           
               
               
           
         
         wherein Nu stands for a nucleotide having a sugar moiety which may be modified, the numbers below the nucleotide sequence designate the position of the nucleotides adjacent to the central nucleotide of the Central Base Triplet having the number 0 whereby the negative numbers designate the 5′ end and the positive number designate the 3′ end of the oligonucleotide and wherein a-j designate the nature of the linkage between the single nucleotides whereby at least linkages a, d, and e are phosphorothioate linkages and whereby at least 2 linkages are a phosphate linkage(s). 
       
     
     
         2 . A chemically modified oligonucleotide according to  claim 1  characterized in that the linkages h and i are phosphate linkages. 
     
     
         3 . A chemically modified oligonucleotide according to  claim 1  characterized in that also the linkage f is a phosphorothioate linkage. 
     
     
         4 . A chemically modified oligonucleotide according to  claim 1  characterized in that also the linkage j is a phosphorothioate linkage. 
     
     
         5 . A chemically modified oligonucleotide according to  claim 1  characterized in that also the linkage g is a phosphorothioate linkage. 
     
     
         6 . A chemically modified oligonucleotide according to  claim 1  characterized in that also the linkages f and j are phosphorothioate linkages. 
     
     
         7 . A chemically modified oligonucleotide according to  claim 1  characterized in that also the linkages b and/or c are phosphorothioate linkages. 
     
     
         8 . A chemically modified oligonucleotide according to  claim 1  characterized in that at least one linkage is a stereopure phosphorothioate linkage. 
     
     
         9 . A chemically modified oligoribonucleotide according to  claim 1  characterized in that
 a) at least 90% of the pyrimidine nucleosides outside the Central Base Triplet are chemically modified, either at the 2′ position of the sugar moiety, or are deoxyribonucleosides, or a combination thereof, 
 b) no more than 6 consecutive nucleosides are chemically modified with 2″-O-methyl at the 2″ position of the sugar moiety, 
 c) at least two of the three nucleosides of the Central Base Triplet are chemically modified at the 2′ position of the sugar moiety, or are deoxyribonucleosides, or a combination thereof. 
 
     
     
         10 . A chemically modified oligonucleotide according to  claim 1  characterized in that at least 50%, more preferably at least 80% of the nucleotides from Nu −5 to +5 are modified independently from another by one of the following modifications at the 2″ position of the ribose: 2″-Fluoro, or 2″-O-methyl, or 2″-H (desoxy). 
     
     
         11 . A chemically modified oligonucleotide according to  claim 1  characterized in that all three nucleotides Nu −1, 0, and +1 have an H residue at the 2″ position of the ribose. 
     
     
         12 . A chemically modified oligonucleotide according to  claim 1  characterized in that the nucleotide Nu +1 carries an N-heterocyclic base, preferably a purine derivative, more preferably a hypoxanthine base, or a derivative thereof. 
     
     
         13 . A chemically modified oligonucleotide according to  claim 1  characterized in that the nucleotide Nu 0 carries a nucleobase based on an N-heterocycle, preferably a pyrimidine or pyridine, more preferably a cytosine, or a derivative thereof. 
     
     
         14 . A chemically modified oligonucleotide according to  claim 1  characterized that it has a length of 20 to 80 nucleotides. 
     
     
         15 . A chemically modified oligonucleotide according to  claim 1  characterized that it has a length of 30 to 60 nucleotides. 
     
     
         16 . A chemically modified oligonucleotide according to  claim 1 , wherein, within the core sequence of formula I, only a, d, e and optionally up to three linkages selected from b, c, f, g and j, are phosphorothioate linkages. 
     
     
         17 . A chemically modified oligonucleotide according to  claim 16 , wherein within the core sequence of formula I, only a, d, e and f are phosphorothioate linkages.